(1) Field of Invention
This invention relates to the field of nucleic acid chemistry, more specifically to nucleotide analogs, and still more specifically to “non-standard” nucleotide analogs that, when incorporated into oligonucleotides (DNA or RNA, collectively xNA), present to a complementary strand in a Watson-Crick pairing geometry a pattern of hydrogen bonds that is different from the pattern presented by adenine, guanine, cytosine, and uracil. Most specifically, this invention combines inventive steps that enable the preparation of function oligonucleotides containing non-standard nucleotides that bind to target molecules (called “aptamers”) or catalyze reactions (called “xNAzymes”) by a process of “in vitro selection” (or IVS). IVS is sometimes also called “SELEX”. Most specifically, this invention claims processes that comprise the creation of xNA libraries, selecting from those libraries individual xNA molecules that perform the preselected function to generate a fraction of xNA molecules having enhanced performance capabilities, PCR amplifying these with less than 5% loss of the non-standard nucleotide, and determining the sequence of certain of those performing molecules
(2) Description of Related Art
For two decades, many have sought processes that mimic, in the laboratory, biological evolution to select or evolve DNA or RNA (collectively xNA) molecules that act as ligands, receptors, or catalysts. This process has been called Systematic Evolution of Ligands by Exponential Enrichment (SELEX), “in vitro selection”, or in vitro evolution (collectively referred to as IVS). The xNA ligands and receptors that bind to a preselected target are called aptamers. xNA molecules that catalyze a preselected reaction are called xNAzymes.
The literature describing the history of development of IVS is summarized in the U.S. patent application having a Ser. No. 13/493172 and the title “In vitro selection with expanded genetic alphabets”, which was filed on Jun. 11, 2012, of which this application is a continuation-in-part, and which is incorporated in its entirety by reference.
As generally practiced, IVS generates aptamers or xNAzymes by the following steps:
(a) A library of nucleic acid (xNA) molecules (typically 1014 to 1014 different species) is obtained.
(b) The library is then fractionated to create a fraction that contains molecules better able bind to the preselected target(s), or catalyze the preselected reaction(s), than molecules in the fractions left behind. For example, to generate aptamers, this separation can be done by contacting the library with a solid support carrying the target, washing from the support xNA molecules that do not bind, and recovering from the support xNA molecules that have bound. xNA molecules within the library that bind to the target are said to survive the selection.
(c) The surviving xNAs are then used as templates for the polymerase chain reaction (PCR) process. A low level of mutation may be included in the PCR amplification, creating Darwinian “variation” in an in vitro evolution process.
(d) While it is conceivable that aptamers/xNAzymes having useful binding/catalytic power may emerge in the first “round” of selection, they generally do not. When they do not, the cycle is repeated. With each cycle of fractionation/selection and PCR amplification, the resulting fraction of xNA molecules becomes more enriched in those that bind to the preselected target or catalyze the preselected reaction.
(e) The product xNA aptamer(s) and xNAzyme(s) might be useful if their sequences are not known. However, the utility of these products is nearly always enhanced if their sequences are known, as this allows them to be generated separately. To obtain those sequences, standard IVS procedures generally clone the xNA products in their DNA form (either directly for DNA products, or after conversion to a DNA sequence using reverse transcriptase for RNA products) followed by classical sequencing. Alternatively, next generation sequence can be applied to the mixture of survivors. The elements of this approach are reviewed in U.S. Ser. No. 13/493172.
U.S. Ser. No. 13/493172 also summarizes the many advantages that were anticipated when xNA molecules replace protein molecules have also been realized. U.S. Ser. No. 13/493172 also summarizes the disadvantages of IVS technology, where the outcome has often been disappointing.
In retrospect, this disappointing outcome might be viewed as unsurprising. Proteins are built from 20 different amino acid building blocks that carry much chemical functionality, including positively charged nitrogens on lysine and arginine, general acid-base functionality on histidine, hydrophobic groups on leucine and others, polarizable binding groups (as on tryptophan and methionine), metal coordinating groups (cysteine, histidine, and others), and so on. Structural biology and mechanistic biochemistry identifies roles for all of these in the binding between proteins and their ligands. In contrast, nucleic acids carry little of this functionality.
Further, with only four building blocks, nucleic acids have fewer motifs for folding than proteins. For example, a G-rich region might lead to a particular “G-quartet”, desired to form a specific binding site for a particular target. However, this quartet might be in equilibrium with an alternative folding motif based on G's elsewhere in a sequence involving G:C pairing. The alternative fold need not have any affinity for a target. There are only a limited numbers of interaction types that can be achieved in DNA with just four letters. Further, with low information density arising from four different building blocks, it is difficult to obtain unambiguous folds from standard xNAs. Further, even if the desired fold is the thermodynamic minimum, it can be kinetically slow to achieve, again because of the low information density in standard xNA.
U.S. Ser. No. 13/493172 also reviews the many attempts to improve IVS with functionalized natural DNA and RNA. However, simply functionalizing standard xNA nucleotides (as in SOMAmers) does not greatly expand its diversity of folds. Nor does it increase the information density of the biopolymer. Further, functionalizing GACT encounters a new set of problems. For example, an xNA molecule having a fluorescent group attached to each nucleobase are hard to make using xNA polymerases. Further, in ways that are not fully understood, having each nucleobase carry a functional group can cause the DNA to cease to follow “rule based” molecular recognition essential for its genetic roles.
U.S. Ser. No. 13/493172 also noted how disadvantages of standard IVS might be mitigated by expanding the number of nucleotides in DNA. For example, rearranging hydrogen bond donor and acceptor groups on the nucleobases increases the number of independently replicable nucleosides in DNA and RNA from four to twelve (FIG. 1). In this “artificially expanded genetic information system” (AEGIS), 12 different nucleotide “letters” pair via six distinguishable hydrogen bonding patterns to give a system that can, in principle, pair, be copied, and evolve like natural DNA, but with higher information density and more functional group diversity.
The potential for using AEGIS to support IVS has been recognized since the proposal of the first AEGIS. Indeed, processes for doing IVS with certain AEGIS-containing nucleotides were claimed by U.S. Pat. No. 5,965,363. However, efforts to implement the process disclosed in that patent have failed. Steps (a) and (b) (above) in the IVS process were possible. Libraries of xNA molecules containing AEGIS components could be prepared, Step (a), and these libraries could be fractionated (Step (b)). However, as discussed in U.S. Ser. No. 13/493172, polymerases were not available to perform PCR on DNA molecules containing multiple AEGIS nucleotides. Further, even after polymerases that copied AEGIS nucleotides were obtained, repeated PCR cycling saw their loss, by perhaps as much as 5% loss per cycle seen when isoguanosine was used to implement the puDDA hydrogen bonding pattern. Efforts to prevent their loss led to DNA molecules with multiple sulfur atoms, undesirable for many applications. Still other AEGIS components suffered epimerization, which prevented their being routinely copied.
Further, even if components in a library of AEGIS-containing oligonucleotides could be amplified and the AEGIS components retained, no downstream tools were available to clone the AEGIS-containing xNA aptamers or xNAzymes. Bacteria were not known to accept AEGIS components Further, no process was available to sequence AEGIS-containing xNA aptamers. After many years of attempting to do IVS based on libraries of AEGIS-containing oligonucleotides, it is clear that any claims covering an AEGIS-based IVS in the prior art were not enabled. This specifically includes the process claimed by U.S. Pat. No. 5,965,363. complement “standard base pairs”. Other hydrogen bonding patterns are said to be “non-standard”, and to form with their appropriate complement “non-standard base pairs”.
Relevant Prior Art
IVS processes with nucleotides that implement standard hydrogen bonding patterns have been known for many decades (see references above). From this art, those of ordinary skill might also be able to perform several of the steps of an IVS process for DNA that contains non-standard nucleotides as well, specifically:
(a) The art does teach an ordinarily skilled artisan how to obtain a library of nucleic acid (xNA) molecules incorporating nucleotides carrying various non-standard nucleobases, such as Z, P, 5-methyl-isoC, isoG, and various analogs of isoG, including B. Phosphoramidites suitably protected to support solid phase synthesis of these are known in the art (see U.S. Ser. No. 13/493172). Several are commercially available. Solid phase synthesis of libraries of DNA molecules is likewise known, involving the use of mixtures of phosphoramidites or split-and-pool synthesis. Libraries of RNA molecules can be obtained by transcribing libraries of encoding DNA molecules.
(b) The art does teach an ordinarily skilled artisan how to fractionate the library to separate molecules that bind preselected target(s), or catalyze preselected reaction(s), from molecules that do not. Fractionation for IVS with non-standard nucleotides is not materially different from that used in standard IVS. Further, a variety of variants of selection processes, and various applications of the derived species have been covered by various patents, including:
U.S. Pat. No. 8,071,737: Nucleic acid ligand complexes. This invention covers a method for preparing a therapeutic or diagnostic complex comprised of a nucleic acid ligand and a lipophilic compound or non-immunogenic, high molecular weight compound
U.S. Pat. No. 7,964,356: Method for generating aptamers with improved off-rates. This invention covers methods for producing aptamers and photoaptamers having slower dissociation rate constants than are obtained using SELEX and photoSELEX methods.
U.S. Pat. No. 7,947,447: Method for generating aptamers with improved off-rates. This invention covers improved SELEX methods for producing aptamers that are capable of binding to target molecules and improved photo-SELEX methods for producing photoreactive aptamers.
U.S. Pat. No. 7,709,192: Nucleic acid ligand diagnostic biochip. This invention covers nucleic acid ligand “biochips”, consisting of a solid support to which one or more specific nucleic acid ligands is attached in a spatially defined manner.
U.S. Pat. No. 7,629,151: Method and apparatus for the automated generation of nucleic acid ligands. This invention covers a method and device for performing automated SELEX.
U.S. Pat. No. 7,368,236: Methods of producing nucleic acid ligands. This invention covers methods for the identification and production of improved nucleic acid ligands based on the SELEX process.
U.S. Pat. No. 7,176,295: Systematic evolution of ligands by exponential enrichment: blended SELEX. This invention covers a method for generating blended nucleic acid ligands containing non-nucleic acid functional units.
U.S. Pat. No. 6,933,116: Nucleic acid ligand binding site identification. This invention covers a nucleic acid ligand for use as a diagnostic reagent for detecting the presence or absence of a target molecule in a sample, and a diagnostic reagent.
U.S. Pat. No. 6,855,496: Truncation SELEX method. This invention covers a method for identifying nucleic acid ligands by the SELEX method wherein the participation of fixed sequences is eliminated or minimized.
U.S. Pat. No. 6,730,482: Modified SELEX processes without purified protein. This invention covers a method for obtaining nucleic acid ligands against target proteins without directly purifying the target proteins.
U.S. Pat. No. 6,716,583: Methods of producing nucleic acid ligands. This invention covers methods for the identification and production of improved nucleic acid ligands based on the SELEX process.
U.S. Pat. No. 6,716,580: Method for the automated generation of nucleic acid ligands
This invention covers a method and device for performing automated SELEX.
U.S. Pat. No. 6,706,482: Conditional-SELEX
This invention covers a method for producing nucleic acid ligands that generate a signal, or cause a decrease in the level of a signal, in the presence of a target molecule
U.S. Pat. No. 6,613,526: Systematic evolution of ligands by exponential enrichment: tissue selex
This invention covers methods to create high-affinity oligonucleotide ligands to complex tissue targets, specifically nucleic acid ligands having the ability to bind to complex tissue targets,
Brief Summary of the Invention
This invention provides processes to generate aptamers and xNAzymes that contain nonstandard nucleotide components by in vitro selection (IVS) methods. Specifically, the invention enables steps essential for IVS that have previously not been enabled for xNA molecules containing nonstandard nucleotides: (i) their PCR amplification and (ii) their sequencing. More specifically, this invention generates aptamers and xNAzymes from the nonstandard nucleotides 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)one (trivially called dP), 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone (trivially called dZ), and nucleotide analogs carrying the 7-deazaisoguanine (trivially called dB), and isocytosine heterocycles.